The role of Vitamin D metabolism in Non- Small Cell Lung Cancer This proposal aims to study local tissue regulation of 1, 25 (OH)2 D3, the active form of Vitamin D in lung cancer. Our preliminary data have shown a differential expression of CYP24A1 (the major catabolizing enzyme for 1, 25 (OH)2 D3 in lung cancer versus normal lung and that high levels of CYP24A1 confer adverse prognosis in early lung cancer. It is our hypothesis that high levels of CYP24A1 in lung cancers will abrogate the antiproliferative and differentiative effects of 1, 25 (OH)2 D3. In the first aim, we will use in vitro studies to study the functional significance of high CYP24A1 in lung cancer cell lines. In the second aim, we will compare the biological aggressiveness and metastatic potential of a murine lung cancer cell line LLC that has been genetically manipulated to express high or low CYP24A1 in a mouse lung cancer model. In the third aim, we will prospectively study the significance of high CYP24A1 expression in patients with advanced non- small cell lung cancer. For the first aim, we will use proliferation assays, apoptotic markers, mass spectrophotometry and CYP24A1 blocking agents in cell lines that have either high or low CYP24A1. Cellular proliferation, apoptosis and 1, 24, 25-dihydroxy D3 (a metabolite of 1, 25 (OH)2D3) will be measured as readouts of CYP24A1 functional activity. For the second aim, we will use stable LLC transfectants that express either high or low CYP24A1 in a murine model of lung cancer metastasis. After establishing the role of tumor-specific CYP24A1 expression, we will treat mice with exogenous 1, 25 (OH)2 D3 to determine its therapeutic role in tumors with either high or low CYP24A1 expression. For the third aim, we will prospectively study the expression of CYP24A1 in both lung tumor and unaffected respiratory epithelium in patients undergoing bronchoscopy for diagnosis of late stage lung cancer. We will determine the relationship between Vitamin D receptor (VDR)/CYP24A1 gene expression and median survival (primary outcome). Secondarily, we will determine the relationship between Vitamin D receptor (VDR)/CYP24A1 gene expression and cancer stage, smoking status and serum 1,25 (OH)2 D3 levels. PUBLIC HEALTH RELEVANCE: The role of vitamin D metabolism in non- small cell lung cancer: relevance to Veterans health. Cancer affects 175,000 American war veterans annually. Lung cancer is the leading cause of cancer death in Veterans. Over 70% of lung cancers are advanced stage at presentation and have a median survival less than 1 year. The goal of this project is to provide the veterans, and society at large a novel way of predicting the anti-cancer properties of Vitamin D, by studying the enzyme CYP24A1 that inactivates it. This enzyme is expressed several fold higher in human lung cancer compared with normal lung. It may be necessary to determine tumor-specific CYP24A1 gene expression to tailor vitamin D therapy in individual patients, either by itself or in combination with a CYP24A1 inhibitor. This will have a significant impact on the use of Vitamin D as an adjunct in advanced lung cancer or following resection of early lung cancer to decrease recurrence.